A Preliminary Investigation into Retrospective Calculation of In-Vivo Drug Concentrations in Dried...

A Preliminary Investigation into Retrospective Calculation of In-Vivo Drug

Concentrations in Dried Crime Scene Blood.

Dwain C. Fuller*, Patricia Pisana, Veterans Affairs North Texas Health

Care System, Dallas, TX, U.S.A.

Why?

• Evaluating injured drivers who may have fled the scene of an accident.

• Evaluating whether a wounded aggressor who has fled the scene was possibly drug-impaired.

• Because law enforcement asks.

Analytical Obstacles

• What was the original blood volume?

• Are drugs stable in dried blood?

• Can one adequately recover drugs from dried blood?

Approach

• Phase 1– Determine if the original volume of blood can be

calculated from the weight of its dried residue.

• Phase 2– Determine the stability and recovery of some

representative drugs from a dried blood matrix.

Phase 1 – Weight/Volume Ratio

• Selected 50 random blood specimens submitted for CBC analysis.– CBC’s selected to avoid any particular disease

state– CBC’s are submitted in 5 mL purple-top tubes

containing an average of 7.4 mg EDTA as an anticoagulant, but no preservative

– Each tube contained 3.5 - 4 mL blood

Phase 1 – Weight/Volume Ratio

• Demographics– Gender: Males – 48, Females – 2– Age: Mean – 63 (33-90)– Race: • Caucasian – 28• African American – 13• Hispanic – 3• Unknown - 6

Phase 1 – Weight/Volume Ratio

• Specimens were mixed for at least 15 min.• Weighing boat weighed and weight recorded.• 500 µL of blood was pipetted into weighing

boat.• Total weight was immediately recorded.• Boat was dried for at least 72 hours at room

conditions (71 ±1⁰F, Relative humidity: 42 ±8%)

Phase 1 – Weight/Volume Ratio

• Boat with dried blood was re-weighed and weight recorded.

• Weight of dried blood per milliliter of liquid blood was calculated.

Other considerations

• Weight of dried blood was corrected for weight of EDTA anticoagulant (2 mg/mL).

• One analyst performed all pipetting to avoid interindividual differences in technique.

• A few specimens were subjected to a 60⁰C oven for an additional 24 hours and re-weighed to determine if drying conditions were adequate.

Results

Mean: g dried blood/mL liquid blood =

0.215 ± 0.025 g (0.161 - 0.259)CV = 11.6 %

Prediction Uncertainty: approximately ± 23% with

95% confidence

Also calculated:

• Blood density

• % Water

Comparison to other studies

Density:

1.052 ± 0.020 g/mL Present Study

1.055 g/mL (Karch, Forensic Issues in Alcohol Testing, CRC Press, 2007)

1.06 g/mL (Cutnell, et al. Physics, 4th edition, Wiley, 1998)

1.025 – 1.125 g/mL (Benson, Katherine, MCAT Review, Emory University,1999)

1.043 – 1.057 g/mL (Hinghofer-Szalkay, et al., Continous Monitoring of blood volume changes in Humans, Journal of Applied Physiology, Vol 63, 1987)

Comparison to other studies

Percent water:79.6 ± 2.4 % g/g Present Study

76.9 – 82.0 % g/g80.5 % g/g – Females78.9 % g/g – Males

Lijnema, et al., Gravimetric determination of the water concentration in whole blood, plasma and erythrocytes and correlations with hematological and clinicochemical parameters. Clinica Chimica Acta, 214, 1993

Limitations of study

• Sample size• Age• Gender• Race• Disease states (diabetes, etc.)• EDTA blood may not be representative of

crime scene blood

Phase 2 – Drug Stability and Recovery

• Pooled EDTA blood and screened for drugs of interest

• Spiked blood with amphetamine, methamphetamine, MDA, MDMA, codeine, morphine, hydrocodone, hydromorphone, cocaine, ecgonine methyl ester, and benzoylecgonine

• Determined original concentration

Phase 2 – Drug Stability and Recovery

• Prepared 1 mL aliquots in weighing boats and dried as previously described

• Analyzed for target drugs as convenient over a period of weeks and months

• Compared results of dried specimens converted the concentration to the original liquid blood concentration

Brief Analytical Method

• Dried blood specimen was transferred into a disposable 16 x 100 mm culture tube.

• Dried blood specimen was ground to a powder using a 10 mm diameter glass stir rod.

• Powdered dried blood was transferred to a tared tube for analysis and weight was recorded.

Brief Analytical Method

• Reconstitute dried blood with buffer used for extraction either liquid:liquid or SPE.

• Add appropriate internal standard(s).• Rotate on laboratory rotator for 10 minutes.• Shake vigorously, (centrifuge if proceeding

with SPE extraction.)• Proceed with extraction as normal.

Recovery(Compared to original spiked blood)

Amphetamine 95 %Methamphetamine 93 %

MDA 94 %

MDMA 85 %

Codeine 96 %

Morphine 99 %

Hydrocodone 95 %

Hydromorphone 94 %

Cocaine 84 % *

Ecgonine methyl ester 103 % *

Benzoylecgonine 98%

*These values may reflect some breakdown of cocaine to EME

Amphetamine

0 20 40 60 80 100 120 140 160 180 200-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [amphetamine] vs. Original Liquid Specimen

Day

Perc

ent

Methamphetamine

0 20 40 60 80 100 120 140 160 180 200-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [Methamphetamine ] vs. Original Liquid Specimen

Day

Perc

ent

MDA

0 20 40 60 80 100 120 140 160 180 200-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [MDA] vs. Original Liquid Specimen

Day

Perc

ent

MDMA

0 50 100 150 200-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [MDMA] vs. Original Liquid Specimen

Day

Perc

ent

Codeine

0 20 40 60 80 100 120 140 160 180-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [Codeine] vs. Original Liquid Specimen

Day

Perc

ent

Morphine

0 20 40 60 80 100 120 140 160 180-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [Morphine] vs. Original Liquid Specimen

Day

Perc

ent

Hydrocodone

0 20 40 60 80 100 120 140 160 180-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [Hydrocodone] vs. Original Liquid Specimen

Day

Perc

ent

Hydromorphone

0 20 40 60 80 100 120 140 160 180-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%% [Hydromorphone] vs. Original Liquid Specimen

Day

Perc

ent

Cocaine studies

Because of expected lability of cocaine and its metabolites, their stability was determined by comparison to a freshly spiked liquid blood specimen using the original spiking solution stored at -80⁰C, rather than the original liquid blood specimen itself.

Cocaine

0 50 100 150 200 250-80%

-60%

-40%

-20%

0%

20%

40%

60%

80%

% [Cocaine] vs. Spiked Standard

Day

Perc

ent

Benzoylecgonine

0 50 100 150 200 250-40%

-30%

-20%

-10%

0%

10%

20%

30%

40%

% [Benzoylecgonine] vs. Spiked Standard

Day

Perc

ent

Ecgonine Methyl Ester

0 50 100 150 200 250-80%-70%-60%-50%-40%-30%-20%-10%

0%10%20%

% [Ecgonine Methyl Ester] vs Spiked Standard

Day

Perc

ent

Observations on cocaine stability in dried blood

Benzoylecgonine is considerably more stable in dried blood at room temperature than is EME.

Isenschmid, Levine, Caplan. JAT, 13:250; 1989

Observations on cocaine stability in dried blood

Benzoylecgonine probably breaks down to ecgonine. Thus ecgonine would probably be an important target analyte to demonstrate cocaine use when analyzing dried blood.

Logan. JAT, 25:219; 2001Skopp, Klingman, et al. Ther. Drug. Monit. 23:174; 2001

A special note about cocaine stability in dried blood.

• Present study– Loss of 44% of cocaine in dried blood in 14 days

• Alfazil and Anderson, JAT, 32:511; 2008– Loss of 19.9% of cocaine in dried blood spots on

filter paper in one month at room temperature– blood matrix was prepared by diluting packed

cells with isotonic saline

A special note about cocaine stability in dried blood. (cont.)

• Baselt, Journal of Chromatography, 268, 1983, 502-505– Cocaine loss in unpreserved plasma was more rapid

than that of unpreserved whole blood– This may reflective of a higher pseudocholinesterase

concentration in plasma compared to whole blood.• Thus Alfazil and Anderson’s results may be

skewed due to their choice of matrix.

Cocaine Stability – Liquid vs. Dried Blood

0 50 100 150 200 250-100%

-80%

-60%

-40%

-20%

0%

20%

% Diff Dried blood% Diff liquid Blood

Day

BE Stability – Liquid vs. Dried Blood

0 50 100 150 200 250-80%

-70%

-60%

-50%

-40%

-30%

-20%

-10%

0%

10%

% Diff Dried Blood% Diff Liquid Blood

Day

EME – Liquid vs. Dried Blood

0 50 100 150 200 250-100%

-80%

-60%

-40%

-20%

0%

20%

40%

60%

% Diff Dried blood% Diff liquid Blood

Day

Observations of Cocaine stability in dried blood vs. liquid blood

• Both cocaine and BE appear to be more stable in unpreserved dried blood than liquid blood.

• Cocaine appears to degrade more to BE rather than EME in unpreserved dried blood as compared to liquid blood.

Limitations of Study

• Spiked EDTA blood may not be representative of crime scene blood– Clotting – Protein binding– Issues regarding removal dried blood from

differing substrates• Actual crime scene blood may be subjected to

a wide array of temperatures and weather conditions

Future studies

• Evaluate larger more diverse population• Evaluate cocaine and metabolite

concentration changes in the short term• Evaluate more drugs• Evaluate authentic blood specimens– Logistically difficult– Postmortem specimens at autopsy?

• ??????

Conclusions

It may be possible to quantitate drugs in unpreserved dried blood obtained from a crime scene, convert the drug concentrations to their previous liquid blood equivalent, and accurately assess the uncertainty of this process.

Questions